Inflammatory bowel
disease (IBD), ulcerative colitis (UC) and Crohn’s disease (CD) are
characterized by ongoing mucosal inflammation in which dysfunction of
the host immunologic response against dietary factors and commensal
bacteria is involved. The chronic inflammatory process leads to
disruption of the epithelial barrier, and the formation of epithelial
ulceration. This permits easy access for the luminal microbiota and
dietary antigens to cells resident in the lamina propria, and stimulates
further pathological immune cell responses. Cytokines are essential
mediators of the interactions between activated immune cells and
non-immune cells, including epithelial and mesenchymal cells. The
clinical efficacy of targeting TNF-a
clearly indicates that cytokines are the therapeutic targets in IBD
patients. In this manuscript, we focus on the biological activities of
recently-reported cytokines [Interleukin (IL)-17 cytokine family, IL-31
and IL-32], which might play a role through interaction with TNF-a
in the pathophysiology of IBD.

Inflammatory bowel
diseases (IBD), ulcerative colitis (UC) and Crohn’s disease (CD) are
chronic intestinal disorders of unknown etiology[1,2]. The
most widely accepted hypothesis on the pathogenesis of IBD is that the
mucosal immune system shows an aberrant response towards luminal
antigens such as dietary factors and/or commensal bacteria in
genetically susceptible individuals[1,3,4]. Environmental
factors may also precipitate the onset or reactivation of this disease[1,3,4].
The chronic inflammatory process leads to disruption of the epithelial
barrier, and the formation of epithelial ulceration. Abnormal bacterial
killing based on genetic factors such as the NOD2 gene mutation
also induces mucosal damage. The easy access for the luminal microbiota
and dietary antigens into cells resident in the lamina propria thus
stimulates pathological immune cell responses.

Cytokines are essential mediators of the
interactions between activated immune cells and non-immune cells,
including epithelial and mesenchymal cells[5]. The clinical
efficacy of targeting TNF-a
clearly indicates that cytokines are one of the therapeutic targets of
chronic inflammatory disorders such as IBD. Infliximab, a mouse/human
chimeric mAb that binds to TNF-a,
has shown efficacy in the treatment of moderate-to severe CD and a
number of CD-related complications[6-8]. Although the precise
mechanism of the action of infliximab is not clear, it neutralizes
soluble TNF-a,
alters cell signaling, and induces the apoptosis of activated
inflammatory cells expressing surface TNF-avia multiple
pathways. On the other hand, some patients are resistant to anti-TNF-a
therapy. In such patients, the disease might be driven by molecular
mechanisms dependent on other cytokines distinct from TNF-a.
In this manuscript, we focused on recently-reported cytokines
[Interleukin (IL)-17 cytokine family, IL-31 and IL-32] which might play
a role through interactions with TNF-a
in the pathophysiology of IBD.

IL-17 CYTOKINE FAMILY

IL-17 family and
Th17 cells

The interleukin (IL)-17
cytokine family is a group of T cell derived cytokines. IL-17A was
originally cloned by Rouvier et al[9] and named CTLA8.
It was subsequently renamed IL-17, and was more recently termed IL-17A.
IL-17A stimulates various cell types to secrete various cytokines and
chemokines, resulting in the induction of inflammation[10-15].
The IL-17 family may play a role in a number of diseases mediated by
abnormal immune responses, such as rheumatoid arthritis[16,17],
pulmonary disease[15,18], lupus[19], multiple
sclerosis[20], and IBD[21]. Homology-based cloning
recently revealed five additional members of the IL-17 family, termed
IL-17B to IL-17F[10]. Among the IL-17 family members, IL-17F
has the highest degree of homology with IL-17A (40% to 55%), followed by
IL-17B (29%), IL-17D (25%), IL-17C (23%), and IL-17E (also named IL-25)
is the most distantly related (17%)[10]. The major cellular
source of IL-17A was initially described as activated CD4+ memory T
lymphocytes. But, it was subsequently demonstrated that CD8+ memory T
lymphocytes, eosinophils, neutrophils and monocytes can also produce
IL-17A[10,22]. The cellular sources of IL-17B and IL-17C have
not been identified. IL-17D is derived from resting CD4+ T cells and
CD19+ B cells[15]. But, IL-17E (IL-25) expression is
restricted to Th2 cells and mast cells[15]. The cellular
source of the last member, IL-17F, has been reported to be activated
CD4+ T cells, basophils and mast cells[15].

One recent topic in
immunology is the newly identified Th17 lineage of CD4+ T cells[23].
Th17 cells are characterized by the production of a distinct profile of
effector cytokines, including IL-17A, IL-17F, IL-6, IL-22 and IL-26, and
have probably evolved to enhance immune and host defense responses
distinct from those targeted by Th1 and Th2 cells[24-26].
Th17 cells develop from naïve CD4 T cell precursors in the presence of
IL-6 and TGF-b,
and full differentiation to Th17 cells is dependent on IL-23[23].
Recent studies demonstrated a role for IL-21 in Th17 development[24,27].
IL-21 serves as an autocrine factor secreted by Th17 cells that promotes
or sustains the Th17 lineage commitment. On the other hand, Th1 cells
develop from naïve CD4 T cell precursors in the presence of IFN-g,
whereas Th2 cells develop under the control of IL-4. Both IFN-g
and IL-4 inhibit Th17 cell proliferation[28]. A recent study
showed that the proliferation of Th17 cells is also inhibited by IL-27,
an IL-12-related cytokine frequently present at sites of inflammation[29].
Th17 cells are characterized as a source of IL-17A and IL-17F, and much
attention has been focused on their functions in normal and pathological
immune responses.

We previously
demonstrated that IL-17A-positive cells were increased in the inflamed
mucosa of IBD patients[21], and a recent study showed that
IL-17F mRNA expression in the mucosa was elevated in CD patients[30].
These observations suggest that IL-17F as well as IL-17A might play a
role in the inflammatory responses involved in the pathophysiology of
IBD.

Inflammatory
responses induced by IL-17A and IL-17F

IL-17A promotes the
expansion and recruitment of innate immune cells such as neutrophils,
and also cooperates with TLR ligands, IL-1b,
and TNF-a
to enhance inflammatory reactions, and to stimulate the production of
beta-defensins and other antimicrobial peptides[25,27]. Its
receptor, IL-17RA, is ubiquitously expressed and shares many features
with classical innate immune receptors such as shared intracellular tail
motifs and convergence onto common inflammatory transcription pathways[31].
To investigate the genes altered in response to an IL-17A stimulus, we
performed a cDNA microarray analysis in human colonic subepithelial
myofibroblasts (SEMFs)[32]. Human colonic SEMFs are located
immediately subjacent to the basement membrane in the normal intestinal
mucosa, juxtaposed against the bottom of the epithelial cells[33,34],
and play a role in inflammation and wound healing in the intestine[33-36].
As shown in Table 1, IL-17A up-regulated several genes which have been
reported to exert pro-inflammatory actions in the pathophysiology of
acute and/or chronic inflammation. In particular, the induction of
CXC-chemokines mRNA expression suggests that IL-17A is a potent inducer
of innate immune responses via the accumulation and activation of
neutrophils in the mucosa.

Recently, we found
that among the IL-17 family members, IL-17F also strongly induced the
secretion of inflammatory cytokines (IL-6, IL-8 and LIF) and matrix
metalloproteinases (MMP-1 and MMP-3) in human colonic SEMFs[32].
Like IL-17A[37],
IL-17F stimulated IL-6, IL-8 and MCP-1 secretion via NF-kB
and MAP kinase activation in human colonic SEMFs. The IL-6, IL-8 and LIF
secretion by human SEMFs in response to IL-17F as well as IL-17A
emphasizes the importance of Th17 products in the induction of
intestinal inflammation. Furthermore, the induction of IL-6 secretion
from colonic SEMFs has a particular significance in the regulation of
immune responses, and in the pathophysiology of IBD; IL-6 has recently
been identified as an indispensable factor for the development of Th17
cells[23].
IL-6 commits naïve CD4 T cell precursors to differentiate into Th17
cells[23].
IL-17A and IL-17F might function as potent stimulators for IL-6
production, suggesting an amplification loop for the local development
and maturation of Th17 cells. Therefore, the IL-17-colonic SEMFs-IL-6
axis may be important for local Th17 development in the intestinal
mucosa.

IL-17A/IL-17F
augments TNF-a-induced
inflammatory responses

As more important
observations, IL-17A and IL-17F augment the TNF-a-induced
IL-6 secretion in human colonic SEMFs[32,37].
This augmentation of TNF-a-induced
IL-6 production by IL-17A is mediated by enhanced stability of the IL-6
mRNA[37,38].
A similar molecular mechanism can be postulated for the augmentation by
IL-17F. IL-17A/IL-17F also augments the TNF-a-induced
expression of granulocyte-colony stimulating factor (G-CSF) and
granulocyte/macrophage (GM)-CSF in human colonic SEMFs[39].

To further investigate
the effects of IL-17A/IL-17F on TNF-a-induced
genes, the alterations in gene expression were analyzed by cDNA
microarrays in human colonic myofibroblasts. As shown in Table 2, IL-17A
further enhanced the expression of various TNF-a-induced
genes, such as IL-6, CXC-chemokines and CSFs. These observations suggest
that the interactions between TNF-a
and IL-17A/IL-17F potently mobilized neutrophils, partially through
granulopoiesis and CXC chemokine induction, as well as through increased
survival locally. This interaction also potently stimulated Th17
development through the stimulation of IL-6 secretion. Thus, a
modulation of the immunological functions of colonic SEMFs by
Th17-derived cytokines may be critical for the development of Th17 cells
and the mucosal innate immune responses (Figure
1).

Interactions
between IL-17A and IL-22

IL-22 was
originally described as an IL-9-induced gene, and was termed
“IL-10-related T cell-derived-inducible factor” (IL-TIF)[40,41].
IL-22 has 22% amino acid identity with IL-10, and belongs to a family of
cytokines with limited homology to IL-10, namely IL-19, IL-20, IL-22,
IL-24 and IL-26. The major sources of IL-22 are activated T cells, and
IL-22 expression in other leukocyte populations such as monocytes,
dendritic cells, NK cells and neutrophils is negligible. Recent studies
have shown that Th17 cells are a source of IL-22[42,43].
We recently found that IL-22 expressing cells were increased in the
inflamed mucosa of IBD patients[44].
In SEMFs, IL-22 upregulates the expression of inflammatory genes such as
IL-6, IL-8, IL-11 and LIF via NF-kB,
AP-1 and MAP-kinase dependent pathways[44].
Furthermore, the combination of IL-17A plus IL-22 showed an additive
effect on transcription factor activation. These concerted responses
were also observed as additive effects on cytokine mRNA expression and
protein secretion. Thus, the cooperation between Th17 derived cytokines
such as IL-17A and IL-22 may play an important role in the
pathophysiology of IBD.

IL-31

IL-31 has a Th2 cell origin

IL-31 was cloned,
and then found to be mainly produced by CD4+ T cells[45],
in particular by skin-homing CD45RO+ (memory) T cells. Transgenic mice
overexpressing IL-31 either with a lymphocyte-specific promoter or a
ubiquitous promoter exhibit a skin phenotype closely resembling atopic
dermatitis in human subjects[45].
In these mice, IL-31 seems to be preferentially produced by T cells
skewed towards a Th2 phenotype; however, these Th1-skewed T cells also
produce substantial amounts of IL-31[45].
IL-31 mRNA expression is widely detected in various organs, including
the gastrointestinal tract[45].

IL-31 is most
closely related to the family of IL-6-type cytokines known to be
involved in many immunomodulatory functions, particularly the
acute-phase response, but also in the proliferation of B and T cells[46].
A recent study indicated that IL-31 sustains the survival of
hematopoietic stem cells, and contributes to effects on the cycling and
numbers of hematopoietic stem cells in vivo[47].
However, IL-31 is clearly distinct from the IL-6-type cytokines because
it does not signal through glycoprotein-130 (GP-130), the common
signaling receptor subunit. IL-31 uses an earlier described orphan
receptor, the glycoprotein 130-like monocyte receptor or glycoprotein
130-like receptor (GPL)[48],
in combination with the oncostatin M receptor (OSMR)[45],
which is expressed on epithelial cells and keratinocytes[45,49].

IL-31 binds
directly to the GPL, and OSMR mainly plays a role in delivering the
signaling information into the cells. GPL and OSMR are widely expressed
in various tissues, including the gastrointestinal tract. This suggests
a role for IL-31 in the immune and inflammatory responses of the
intestine. In response to IL-31, its receptor complex recruits the Jak1,
Jak2, STAT-1, STAT-3 and STAT5 signaling pathways, as well as the
PI3-kinase/AKT cascade[49].
SHP-2 and Shc adaptor molecules are also recruited, and contribute to an
increased activation of the MAP kinase pathway in response to IL-31[49].
Despite the extensive study of intracellular signaling pathways
activated by IL-31 stimulation, the cellular responses to IL-31 were
barely investigated in any cell type.

IL-31 stimulates
inflammatory responses in colon myofibroblasts

To define the role of
IL-31 in the intestinal mucosa, we investigated how IL-31 modulates mRNA
expression in human colonic SEMFs. An analysis of the cDNA microarrays
indicated that IL-31 effectively induced the secretion of chemokines
[CXCL8 (IL-8), CXCL1 (growth-related oncogene; GRO-a)],
CCL7 (monocyte chemoattractant protein-3; MCP-3), CXCL3, CCL13, CCL15),
proinflammatory cytokines (IL-6, IL-16 and IL-32), and matrix
metalloproteinases (MMP-1, MMP-3, MMP-25 and MMP-7). The stimulatory
effects of IL-31 were comparable to the effects of IL-17A. Furthermore,
simultaneous stimulation with IL-31 and IL-17A showed additive effects
on IL-6, IL-8, GRO-a,
MCP-3, MMP-1 and MMP-3 secretion. Similar effects for IL-31 have been
reported in bronchial epithelial cells[50].
In bronchial epithelial cells, IL-31 could significantly elevate both
gene and protein expressions of epidermal growth factor (EGF), vascular
endothelial growth factor (VEGF) and monocyte chemoattractant protein-1
(MCP-1/CCL2). The combination of IL-31 with either IL-4 or IL-13 further
enhanced VEGF and CCL2 production. In these cells, IL-31 could activate
p38 MAPK, extracellular signal-regulated kinase (ERK) and c-Jun
N-terminal kinase (JNK).

As mentioned above,
IL-31 is a Th2 type cytokine, and these findings indicate that Th2 cells
may be involved in the immune and inflammatory responses of the
intestinal mucosa through IL-31secretion. Since IL-31 and IL-17A
stimulate the secretion of proinflammatory mediators in an additive
manner, Th2-derived IL-31 and Th17-derived IL-17A cooperate in the
pathophysiology of IBD.

IL-32

IL-32 cytokine family

IL-32 is a
recently described cytokine produced by T lymphocytes, natural killer
cells, monocytes, and epithelial cells[51,52].
IL-32 is a proinflammatory cytokine originallydescribed as a
transcript termed NK4, found in activated naturalkiller (NK)
cells and T lymphocytes[53].
Although IL-32 was first reported as a transcript in IL-2 activated NK
and T cells, it appears that the epithelial cells are the dominant and
widespread source[54].
The gene encoding IL-32 is located on human chromosome 16p13.3, and is
organized into eight exons[55].
There are four splice variants (IL-32a,
IL-32b,
IL-32δ and IL-32g),
and IL-32a
is the most abundant transcript. Of particular importance, IL-32 is
prominently induced by interferon (IFN)-g
in lung epithelial cells and monocytes[51].
IL-32 stimulates the secretion of proinflammatory cytokines and
chemokines such as IL-1b,
TNF-a,
IL-6 and IL-8 by via the activation of NF-kB
and p38 mitogen-activated protein kinases (MAPKs)[51,52].
IL-32 has been implicated in inflammatory disorders such as rheumatoid
arthritis[54,56-58],
mycobacterium tuberculosis infection[59,60],
and IBD[61].

Intracellular
accumulation of IL-32

The amino acid
sequence derived from the initial NK4 cDNA containeda signal
peptide without a transmembrane domain[53,54].
But, thetranscript was never expressed as a recombinant
protein, and was not sequenced.Activated humanT cells
generateIL-32 with a molecular weight of 25 kDa, which on
Western blotting analysisis found in the lysates rather than
the supernatants. Similar findings werereported for 293T
cells transfected with either IL-32g
or IL-32b[62].In human peripheral blood mononuclear cells stimulated withConA, most of the IL-32 was found in the lysates[51].
On the other hand, the overexpressionof IL-32a
or IL-32b
in COS cells resulted in secretedIL-32[51].
It remains unclear which isoformsare secreted from which
particular cell type. Activated T cells and NK cells do not secrete
IL-32,or alternatively, the secreted IL-32 is derived from
apoptoticcells due to the presence of GAPDH in the same
supernatants.These observations suggest that IL-32 is
secreted only as a resultof cell death[63].Recently, we observed that in colon cancer cell lines,
proinflammatory cytokines induce the intracellular accumulation of IL-32a,
but does not induce secretion[61].
Similar results were also observed in myofibroblasts isolated from the
normal human pancreas[64].
Goda et al suggest that there is a role for intracellular IL-32
with cell death, since attenuating intracellular IL-32 levels resulted
in decreased cell death[62].
These results also support the conceptthat high levels of
intracellular IL-32b
may inducecell death. One hypothesis is that the
proinflammatory activity of IL-32 may act upon its release through cell
death (apoptosis).

Molecular
mechanisms regulating IL-32ainduction

Shioya et al
demonstrated that stimulation with IL-1b,
IFN-g
and TNF-a
enhanced IL-32a
mRNA expression in three colon cancer cell lines[61].
TNF-a
was the strongest among them. These factors also induced the
intracellular accumulation of IL-32a.
Since transfection with the mutant form of IkBa
inhibited the effects of both IL-1b
and TNF-a
on IL-32a
mRNA expression, NF-kB
must play a role in IL-1b-
and TNF-a-induced
IL-32a
mRNA expression.

Nishida et al
analyzed IL-32a
expression in non-transformed myofibroblasts derived from the normal
human pancreas[64].
IL-32a
mRNA was weakly expressed without any stimulus, and its expression was
markedly enhanced by IL-1b,
IFN-g
and TNF-a.
IL-1b,
IFN-g
and TNF-a
enhanced the intracellular accumulation of IL-32a
protein. But, IL-32a
was not detected in the supernatants. An inhibitor of
phosphatidylinositol 3-kinase, (LY294002) significantly suppressed the
IL-1b-,
IFN-g-
and TNF-a-induced
IL-32a
mRNA expression, although MAPK inhibitors had no effect. Akt activation
in response to these cytokines was confirmed by Western blotting
analysis. Furthermore, LY294002 suppressed both IL-1b-
and TNF-a-induced
NF-kB
activation, as well as IL-1b-,
TNF-a-
and IFN-g-induced
AP-1 activation. A blockade of NF-kB
and AP-1 activation by an adenovirus expressing a stable mutant form of
IkBa
and a dominant negative mutant of c-Jun markedly suppressed the IL-1b-,
IFN-g-
and/or TNF-a-induced
IL-32a
mRNA expression. Thus, they concluded that IL-32a
mRNA expression was dependent on interactions between the PI3K/Akt-pathway
and the NF-kB/AP-1
system.

IL-32a
and IBD

Shioya et alperformed an immunohistochemical analysis
to evaluate the expression of IL-32a
protein in the mucosa of IBD patients[61].
IL-32a
was weakly immunoexpressed by epithelial cells in the normal colonic
mucosa and samples of ischemic colitis. In contrast, the epithelial
expression of IL-32a
was markedly enhanced in the inflamed region of active UC and CD
patients[61].
In particular, IL-32a
expression tends to increase in samples from active CD patients. IL-32a
expression was barely detectable in leukocytes. Thus, these observations
indicate that epithelial cells are the major expression site for IL-32a
in the intestinal mucosa, and that IL-32a
expression is enhanced in the IBD mucosa.

Netea et al
recently demonstrated that IL-32 augments the production of IL-1b
and IL-6 induced by muramyl dipeptide (MDP), a peptidoglycan fraction of
bacteria, by means of nucleotide-binding oligomerization domain proteins
(NOD1 and NOD2) through a caspase-1-dependent mechanism[52].
NODs are a family of intracytoplasmic bacterial sensors, and the
recognition of bacterial peptidoglycans subsequently induces NF-kB
activation[65].
Mutations in NOD2 have been implicated in the pathogenesis of CD[66,67],
and CD patients homozygous for the frameshift 3020insC mutated allele
have defective responses to MDP in terms of cytokine production[68,69].
Recently, it has been shown that NOD2 mutations in CD patients
potentiate NF-kB
activity and IL-1b
processing[70].
Thus, these findings suggest a pivotal role for IL-32 in the
pathophysiology of IBD, and in particular CD. Since IL-32a
is a proinflammatory cytokine characterized by NF-kB
and p38 MAPK activating activities[51,57]
and because IL-32 acts synergistically with NOD ligands to induce
proinflammatory cytokines[52],
the overexpression of IL-32a
in the IBD mucosa strongly suggests that it plays an important role in
the inflammatory and antibacterial responses involved in the
pathogenesis of IBD.

Recent studies
have focused on the role of innate immunity in the pathogenesis of IBD[4].
The initial step of innate immunity is mediated by the recognition of
pathogen-associated molecular patters (PAMPs) through Toll-like
receptors (TLRs) and NOD proteins (NODs)[71].
TLRs are located mainly on cell-surface membranes, but NODs function as
intracellular recognition systems[65,66].
In human monocytes, IL-32 acts synergistically with NOD specific
peptidoglycans for the release of IL-1b
and IL-6[52].
The synergistic effects of IL-32 and the NOD ligands on cytokine
production is abolished in cells from CD patients bearing the NOD2
frameshift mutation 3020insC, indicating that this synergism between
IL-32 plus MDP depends on NOD2[52].
Interactions between NOD-1 and IL-32 also potentiate proinflammatory
cytokine production[52].
Furthermore, Berrebi et alpreviously reported the overexpression of NOD2 in
infiltrated monocytes and epithelial cells in the IBD mucosa[72].
These observations suggest that overexpressed IL-32 may cause a specific
and excessive stimulation of the NOD pathways, which leads to a marked
amplification in IL-1b
and IL-6 production in the IBD mucosa.

IL-32 was initially
characterized as an inducer of TNF-a
in circulating monocytes[51],
and hence inflammatory responses in the affected mucosa of IBD patients
may be amplified by the consecutive loop of IL-32-induced TNF-a
secretion from monocytes and TNF-a-stimulated
IL-32 secretion from epithelial cells. This loop may be further
amplified by the Th1 cytokine IFN-g.
Previously, it has been reported that TNF-a
and IFN-g
synergistically induced the release of NOD2[73],
which supports the coupled regulation of IL-32a
and NOD2. The coupled regulation of IL-32a
with NOD2 may account for the rapid and efficient induction of innate
immune responses at the intestinal mucosa. Furthermore, these data
suggest that an amelioration of IBD symptoms by TNF-a-targeting
therapies may be partially dependent on interference in the TNF-a-IL-32
loop.

The apoptosis of
IECs is considered a normal biological function to eliminate damaged
epithelial cells, and to restore epithelial cell growth, regulation, and
epithelial integrity[74].
An overexpression of cytoplasmic IL-32a
might account for the induction of apoptosis in damaged epithelial cells
at the inflamed mucosa of IBD patients, leading to an efficient
elimination and the rapid induction of mucosal repair. Apoptosis caused
by accumulated IL-32 can be considered a host defense mechanism against
invading microorganisms, in which damaged epithelial cells are
efficiently eliminated along with the invading microorganisms, and thus
any further invasion of the microorganisms can be blocked.

CONCLUSION

In this review, we have
summarized the newly reported cytokines which may play significant roles
in the pathophysiology of IBD. An augmentation of TNF-a
effects by IL-17A/F and a possible amplifying cascade between TNF-a
and epithelial-derived IL-32 are of particular interest. The clinical
efficacy of TNF-a
blocking may be associated with an interruption of these cascades. The
discovery of new cytokines and the determination of their biological
activities may support the development of a novel therapeutic strategy
for the treatment of IBD patients.